The present invention is directed to particular variants of vascular endothelial cell growth factor (hereinafter sometimes referred to as VEGF) which bind to and occupy cell surface VEGF receptors without inducing a VEGF response, thereby antagonizing the biological activity of the native VEGF protein. The present invention is further directed to methods for preparing such variant VEGF antagonists and to methods, compositions and assays utilizing such variants for producing pharmaceutically active materials having therapeutic and pharmacologic properties that differ from the native VEGF protein.
The two major cellular components of the mammalian vascular system are the endothelial and smooth muscle cells. Endothelial cells form the lining of the inner surface of all blood vessels in the mammal and constitute a non-thrombogenic interface between blood and tissue. Therefore, the proliferation of endothelial cells is,an important component for the development of new capillaries and blood vessels which, in turn, is a necessary process for the growth and/or regeneration of mammalian tissues. One protein that has been shown to play an extremely important role in promoting endothelial cell proliferation and angiogenesis is vascular endothelial cell growth factor (VEGF). VEGF is a heparin-binding endothelial cell-specific growth factor which was originally identified and purified from media conditioned by bovine pituitary follicular or folliculostellate (FS) cells. Ferrara and Henzel, Biochem. Biophys. Res. Comm. 161:851-858 (1989). Naturally-occurring VEGF is a dimeric protein having an apparent molecular mass of about 46 kDa with each subunit having an apparent molecular mass of about 23 kDa. Normal dimerization between individual native VEGF monomers occurs through the formation of disulfide bonds between the cysteine residues located at amino acid position 51 of one monomeric unit bonding to the cysteine residue at amino acid position 60 of another monomeric unit and vice versa. Human VEGF is expressed in a variety of tissues as multiple homodimeric forms (121, 165, 189 and 206 amino acids per monomer), wherein each form arises as a result of alternative splicing of a single RNA transcript. For example, VEGF121 is a soluble mitogen that does not bind heparin whereas the longer forms of VEGF bind heparin with progressively higher affinity.
Biochemical analyses have shown that the native VEGF dimer exhibits a strong mitogenic specificity for vascular endothelial cells. For example, media conditioned by cells transfected by human VEGF cDNA promoted the proliferation of capillary endothelial cells, whereas medium conditioned by control cells did not. Leung et al., Science 246:1306 (1989). Thus, the native VEGF dimer is known to promote vascular endothelial cell proliferation and angiogenesis, a process which involves the formation of new blood vessels from preexisting endothelium. As such, the native VEGF may be useful for the therapeutic treatment of numerous conditions in which a growth-promoting activity on the vascular endothelial cells is important, for example, in ulcers, vascular injuries and myocardial infarction. The endothelial cell proliferative activity of the VEGF dimer is known to be mediated by two high affinity tyrosine kinase receptors, fit-1 (FMS-like tyrosine kinase) and KDR (kinase domain region), which exist only on the surface of vascular endothelial cells. DeVries, et al., Science 225:989-991 (1992) and Terman, et al., Oncogene 6:1677-1683 (1991). As cells become depleted in oxygen, because of trauma and the like, VEGF production increases in such cells, wherein the generated VEGF protein subsequently binds to its respective cell surface receptors in order to signal ultimate biological effect. The signal then increases vascular permeability and the cells divide and expand to form new vascular pathways. Thus, native VEGF functions to induce vascular proliferation through the binding to endothelial cell-specific receptors.
While VEGF-induced vascular endothelial cell proliferation is desirable under certain circumstances, vascular endothelial cell proliferation and angiogenesis are also important components of a variety of diseases and disorders. Such diseases and disorders include tumor growth and metastasis, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, age-related macular degeneration, hemangiomas, immune rejection of transplanted corneal tissue and other tissues, and chronic inflammation. Obviously, in individuals suffering from any of these disorders, one would want to have a means for inhibiting, or at least substantially reducing, the endothelial cell proliferating activity of the native VEGF dimeric protein.
Having an available means for inhibiting native VEGF activity is important for a number of reasons. For example, in the specific case of tumor cell growth, angiogenesis appears to be crucial for the transition from hyperplasia to neoplasia and for providing nourishment to the growing solid tumor. Folkman, et al., Nature 339:58 (1989). Angiogenesis also allows tumors to be in contact with the vascular bed of the host, which may provide a route for metastasis of tumor cells. Evidence for the role of angiogenesis in tumor metastasis is provided, for example, by studies showing a correlation between the number and density of microvessels in histologic sections of invasive human breast carcinoma and actual presence of distant metastasis. Weidner et al., New Engl. J. Med. 324:1 (1991). Thus, one possible mechanism for the effective treatment of neoplastic tumors is to inhibit or substantially reduce the endothelial cell proliferative and angiogenic activity of the native dimeric VEGF protein.
Therefore, in view of the role that VEGF-induced vascular endothelial cell growth and angiogenesis play in many diseases and disorders, it is desirable to have a means for reducing or substantially inhibiting one or more of the biological effects of the native VEGF protein, for example, the mitogenic or angiogenic effect thereof. Thus, the present invention is predicated upon research intended to identify novel VEGF variant polypeptides which are capable of inhibiting one or more of the biological activities of native VEGF. Specifically, the present invention is predicated upon the identification of VEGF variants which are capable of binding to and occupying cell-surface VEGF receptors without inducing a typical VEGF response, thereby effectively reducing or substantially inhibiting the effects of native VEGF. It was postulated that if one could prepare such VEGF variants, one could use such variants in instances of tumor treatment in order to starve the tumors for intended regression.
It was a further object of this research to produce VEGF variants which lose the ability to properly dimerize through the formation of covalent cysteine-cysteine disulfide bonds. Such variants include variant VEGF monomers which lack the ability to dimerize through the formation of cysteine-cysteine disulfide bonds and variant VEGF monomers which may dimerize through the formation of at least one cysteine-cysteine disulffide bond, however, wherein at least one disulfide bond differs from that existing in the native VEGF dimer. Such variants possess the ability to bind to and occupy cell surface VEGF receptors without inducing a VEGF response, thereby competing with native VEGF for binding to the receptors and antagonistically inhibiting the biological activity of the native VEGF dimer.
As further objects, the VEGF variants of the present invention can be employed in assays systems to discover small molecule agonists and antagonists for intended therapeutic use.
The results of the above described research is the subject of the present invention. We herein demonstrate that mutation or modification of the cysteine residues at amino acid positions 51 and/or 60 of the native VEGF amino acid sequence functions to produce VEGF variants which lose the ability to properly dimerize. Specifically, substitution of cysteine at positions 51 and/or 60 with another amino acid or modification of the cysteine at that site prevents the ability of that amino acid to participate in the formation of a disulfide bond. These variants, however, retain the ability to bind to and occupy cell surface VEGF receptors without inducing a VEGF response, thereby effectively inhibiting the biological activity of the native VEGF dimer.
The present invention provides variants of the native VEGF protein which are capable of binding to a VEGF receptor on the surface of vascular endothelial cells, thereby occupying those binding sites and inhibiting the mitogenic, angiogenic or other biological activities of the native VEGF protein. The novel antagonist molecules of the present invention, therefore, are useful for the treatment of diseases or disorders characterized by undesirable excessive vascularization, including by way of example, tumors, and especially solid malignant tumors, rheumatoid arthriUs, psoriasis, atherosclerosis, diabetic and other retinopathies, retrolental fibroplasia, age-related macular degeneration, neovascular glaucoma, hemangiomas, thyroid hyperplasias (including Grave""s disease), corneal and other tissue transplantation, and chronic inflammation. The antagonists of the present invention are also useful for the treatment of diseases or disorders characterized by undesirable vascular permeability, such as edema associated with brain tumors, ascites associated with malignancies, Meigs"" syndrome, lung inflammation, nephrotic syndrome, pericardial effusion (such as that associated with pericarditis) and pleural effusion.
In a preferred embodiment, the variant VEGF polypeptides of the antagonist molecules of the present invention comprise amino acid modifications of at least one cysteine residue present in the native VEGF amino acid sequence wherein modification of that cysteine residue(s) results in the polypeptide being incapable of properly dimerizing with another VEGF polypeptide.
In a particularly preferred embodiment, the cysteine residues of the native VEGF amino acid sequence that are modified are at amino acid positions 51 and/or 60 of the native VEGF amino acid sequence.
The novel VEGF variant polypeptides of the present invention may be recombinantly generated by creating at least one amino acid mutation at a cysteine residue in the native VEGF amino acid sequence such that the variant is incapable of properly dimerizing. Typical mutations include, for example, substitutions, insertions and/or deletions. The cysteine residue(s) of interest may also be chemically modified so as to be incapable of participating in a disulfide bond.
In other embodiments, the present invention is directed to isolated nucleic acid sequences encoding the novel VEGF antagonist molecules of the present invention and replicable expression vectors comprising those nucleic acid sequences.
In still other embodiments, the present invention is directed to host cells which are transfected with the replicable expression vectors of the present invention and are capable of expressing those vectors.
In yet another embodiment, the present invention is directed to a composition for treating indications wherein anti-angiogenesis is desired, such as in arresting tumor growth, comprising a therapeutically effective amount of the antagonist molecule of the present invention compounded with a pharmaceutically acceptable carrier. Another embodiment of the present invention is directed to a method of treating comprising administering a therapeutically effective amount of the above described composition.
Expanding on the basic premise hereof of the discovery and mutagenesis of the native VEGF polypeptide to produce variant VEGF polypeptides, the present invention is directed to all associated embodiments deriving therefro m, including recombinant DNA materials and processes for preparing such variants, materials and information for compounding such variants into pharmaceutically finished form and assays using such variants to screen for candidates that have agonistic or antagonistic properties with respect to the native VEGF polypeptide.